This mentored clinical scientist research career development award is a training program designed to help the candidate achieve his long-term career goal to have an academic career with a focus on the retina. For the current training and future career, Brian Hafler intends to perform research focused on understanding the molecular mechanisms underlying development of the human fovea and translational applications to retinal disease. The fovea is responsible for high acuity central vision, yet the genes that establish this region are unknown. This research proposal will focus on identifying the molecular pathways that generate this region using the chicken as a model system, as it has a central rod-free spot, much like the human fovea. In this research proposal, Dr. Hafler will use new technologies like ATAC-seq to perform open chromatin profiling of cells in the developing chicken rod-free zone. ATAC-seq data will be compared with RNA-seq data, which is in the process of being generated in the Cepko lab to prioritize candidate genes of interest. Immunohistochemistry and in situ hybridization will be performed to assess whether the transcripts associated with regions of open chromatin specifically localize to the rod-free central zone in the chicken embryonic retina. Candidate cis- regulatory modules located in regions of open chromatin will be aligned and assessed for conservation with species that contain a fovea. The most promising sequences will be cloned into reporter constructs and tested for activity in the developing chicken retina using electroporation assays. Transcription factors that bind to the cis-regulatory modules will be identified using electrophoretic mobility shift assays. Immunohistochemistry and in situ hybridization on embryonic human retinas will be used to examine if the transcription factors of interest are conserved during human retinal development. Completion of this work would not only give insight into how the fovea is generated during human development, it will also help with the development of cell replacement therapies for patients with age-related macular degeneration. The candidate has an excellent mentor, Dr. Constance Cepko, who is a leader in the field and is committed to the scientific development and execution of the project. She is the Bullard Professor of Genetics and Ophthalmology at Harvard Medical School and a Howard Hughes Medical Institute Investigator. The environment at Harvard Medical School has a strong and established research program related to retinal development. They will provide necessary facilities for career enhancement to become an independent investigator. There are numerous courses offered, seminars, journal clubs, and presentations with opportunities for intellectual interactions with other research scientists. The experience, knowledge, and skills gained through the research plan and career development activities will carry the candidate forward towards a career as an independent clinician-scientist.